This invention relates to the field of cryopreservation of living cells; and more particularly, a method is provided for the cryopreservation of multicellular organisms.
Cryopreservation in its broadest sense would include preservation at above freezing temperatures, as is commonly done in blood banks. However, in the context of this invention, the meaning of the word is restricted to the preservation of living cells at below freezing temperatures.
The field of cryopreservation had its inception in the early attempts to store blood cells at low temperatures. Experiments directed at preserving blood in the frozen state have been documented as early as 1939. (For an historical survey on the cryopreservation of blood cells, see CRYOBIOLOGY, Vol. 6, No. 5, 1970.) Since the early experiments with blood, researchers have expanded the idea of preserving living cells to various other types. Consequently, with such expansion, the field of cryopreservation has developed as a new technology aimed at the preservation of living cells by storing in the frozen state with subsequent revival.
For the most part, cryopreservation of living cells has dealt with single cells only. As reported by D. G. Whittlingham, S. P. Leibo and P. Mazur of the Biology Division of Oak Ridge National Laboratory in an article in SCIENCE, Vol. 178, October 1972, attempts to freeze multicellular mammalian embryos and revive them to survival have met with limited success. The article "Survival of Mouse Embryos Frozen to -196.degree. and -269.degree. C." by the above named authors was an attempt to investigate the mechanisms of freezing injury to multicellular systems and ascertain the critical cryobiological factors. The experiment was conducted on mouse embryos, and the researchers concluded that the cryobiological factors that might influence the survival of frozen multicellular organisms are primarily the suspending medium, cooling rate, final temperature, and warming rate.
In the method of cryopreservation taught by the article, the freezsing rate for survival of the organism is required to be slow, between 0.3.degree. and 2.degree. C. per minute, and warming rates should be from 4.degree. to 25.degree. C. per minute. Storage of the frozen embryos was at -196.degree. C. for several days. The cryoprotectant used was dimethyl sulfoxide (DMSO).
It has been generally accepted and practiced by workers in the field of cryopreservation of living cells that a slow cooling rate below freezing of about 1.degree. C. per minute is required for survival of the cells. Meryman in Annals of the New York Academy of Sciences, Vol. 85, pp. 503-509 (1960) opined that rapid freezing would not work for animal tissues.
With regard to the suspending medium or cryoprotectant, glycerol and dimethyl sulfoxide (DMSO) have been found to be most effective, with about 5% to 15% concentration being commonly used. (See Paul, Cell and Tissue Culture, 4th Ed., pp. 308-315 (1970)). A third type of cryoprotectant that has been used is polyvinylpyrrolidone (PVP), and a fourth type is dextrose (glucose) and/or sucrose. A discussion of the combined effects of freezing rates and various cryoprotective agents on the preservation of human erythrocytes is presented in CRYOBIOLOGY, Vol. 4, No. 5 (1968) authored by Rapatz and Leyet. One of the summary conclusions drawn by these researchers is that glycerol and DMSO are effective in preventing lysis at low cooling rates, but are injurious at high cooling rates.
Methods of cryopreservation used have generally involved first suspending the cells or organism in a liquid cryoprotectant medium suitable for freezing, usually a solution containing about 5% to about 15% DMSO or glycerol. Next, the cell suspensions are frozen slowly at a rate of about 1.degree. C. per minute and maintained at a storage temperature of -70.degree. C. or lower. For revival, the frozen cell suspension is thawed, and the cells are separated from the cryoprotectant medium.
Specific methods of cryopreservation are presented in U.S. Pat. No. 3,943,993 (Smith) entitled "Low Temperature Storage of Surface Attached Living Cell Cultures," U.S. Pat. No. 3,940,943 (Sikes) entitled "Multistage Freezing System for Preservation of Biological Materials," and U.S. Pat. No. 3,303,662 (Moline) entitled "Process for Cell Preservation."
Smith relates to a process for cooling, storing and reviving surface attached living cell cultures. The process involves preparing an attached cell culture and exposing the cells to a storage medium containing about 10% dimethyl sulfoxide. The temperature is lowered at a rate of 1.degree. C. per minute until about -20.degree. C. or lower is reached. The cell cultures are then transferred to a container having a temperature of about -70.degree. C. or less, where the cells are kept until time to withdraw them for revival. The cells are warmed and the storage medium is replaced by an appropriate cell culture medium.
Sikes relates to a freezing and thawing process for the preservation of animal semen, blood and other biological materials. In the process described, a sample of semen is placed in a cryoprotective agent and cooled slowly from body temperature to +5.degree. C. and held there for 30 minutes or longer. The temperature is rapidly lowered to a temperature slightly below freezing and held for one to eight minutes. The temperature is then taken down to -100.degree. C. at a rate of 20.degree. C. per minute. The sample is then immersed into liquid nitrogen for final cooling and storage. To revive the cells, the sample and protective agent are thawed by immersion in +45.degree. C. water.
The process described in Moline is one for preserving animal organs. In the process, the organ is perfused by a protective agent and cooled to the freezing point. The organ is further cooled through the liquid-solid phase change in less than ten minutes, with cooling of the organ to about -50.degree. C. at a rate of not more than about 3.degree. C. per minute. The organ is further cooled to a temperature at or below -130.degree. C. where it is maintained.
All of the above described methods, though dealing with various types of cellular arrangements, follow a basic theory that cryopreservation techniques must be conducted with a slow cooling rate below the freezing point in order to achieve survival of the cells after thawing. Also, the methods have all used cells or tissues from terrestrial mammals.